Test systems and sensors for detecting molecular binding events

ABSTRACT

A bio-assay test system includes a test fixture, a measurement system, and a computer. The test fixture includes a bio-assay device having a signal path and a retaining structure configured to place a sample containing molecular structures in electromagnetic communication with the signal path. The measurement system is configured to transmit test signals to and to receive test signals from the signal path at one or more predefined frequencies. The computer is configured to control the transmission and reception of the test signals to and from the measurement system.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part application of U.S.application Ser. No. 09/243,194, entitled “Method and Apparatus forDetecting Molecular Binding Events,” filed Feb. 1, 1999, which claimsthe benefit of U.S. Provisional Application No. 60/073,445, entitled“Detection of Molecular Binding Events on a Conductive Surface,” filedFeb. 2, 1998.

[0002] Further, the following applications are herein incorporated byreference in their entirety for all purposes:

[0003] “A Sensitive Detection of Dispersions in Aqueous-based,Surface-bound Macromolecular Structures Using Microwave Spectroscopy,”Ser. No. 60/134,740, filed May 18, 1999;

[0004] “Methods of Nucleic Acid Analysis,” Atty Docket 019501-000600,filed concurrently herewith; and

[0005] Methods for Analyzing Protein Binding Events,” Atty Docket019501-000700, also filed currently herewith.

BACKGROUND

[0006] Virtually every area of the biomedical sciences is in need of asystem to assay chemical and biochemical reactions and determine thepresence and quantity of particular analytes. This need ranges from thebasic science research lab, where biochemical pathways are being mappedout and their functions correlated to disease processes, to clinicaldiagnostics, where patients are routinely monitored for levels ofclinically relevant analytes. Other areas include pharmaceuticalresearch and drug discovery applications, DNA testing, militaryapplications such as biowarfare monitoring, veterinary, food, andenvironmental applications. In all of these cases, the presence andquantity of a specific analyte or group of analytes, needs to bedetermined.

[0007] For analysis in the fields of pharmacology, genetics, chemistry,biochemistry, biotechnology, molecular biology and numerous others, itis often useful to detect the presence of one or more molecularstructures and measure interactions between molecular structures. Themolecular structures of interest typically include, but are not limitedto, cells, antibodies, antigens, metabolites, proteins, drugs, smallmolecules, enzymes, nucleic acids, and other ligands and analytes. Inmedicine, for example, it is very useful to determine the existence of acellular constituents such as receptors or cytokines, or antibodies andantigens which serve as markers for various disease processes, whichexists naturally in physiological fluids or which has been introducedinto the system. In genetic analyses, fragment DNA and RNA sequenceanalysis is very useful in diagnostics, genetic testing and research,agriculture, and pharmaceutical development. Because of the rapidlyadvancing state of molecular cell biology and understanding of normaland diseased systems, there exists an increasing need for methods ofdetection, which do not require labels such as fluorophores orradioisotopes, are quantitative and qualitative, specific to themolecule of interest, highly sensitive and relatively simple toimplement. Many known targets such as orphan drug receptors, and manymore targets becoming available, have no known affinity ligands, so thatunlabeled means of detecting molecular interactions are highlydesirable. In addition, the reagent costs for many labeled assaytechnologies are quite expensive, in addition to the economic andenvironmental costs of disposing of toxic fluorophores andradioisotopes.

[0008] Numerous methodologies have been developed over the years to meetthe demands of these fields, such as Enzyme-Linked Immunosorbent Assays(ELISA), Radio-Immunoassays (RIA), numerous fluorescence assays, massspectroscopy, colorimetric assays, gel electrophoresis, as well as ahost of more specialized assays. Most of these assay techniques requirespecialized preparations, especially attaching a label or greatlypurifying and amplifying the sample to be tested. To detect a bindingevent between a ligand and an antiligand, a detectable signal isrequired which relates to the existence or extension of binding. Usuallythe signal is provided by a label that is conjugated to either theligand or antiligand of interest. Physical or chemical effects whichproduce detectable signals, and for which suitable labels exist, includeradioactivity, fluorescence, chemiluminescence, phosphorescence andenzymatic activity to name a few. The label can then be detected byspectrophotometric, radiometric, or optical tracking methods.Unfortunately, in many cases it is difficult or even impossible to labelone or all of the molecules needed for a particular assay. Also, thepresence of a label may make the molecular recognition between twomolecules not function for many reasons including steric effects. Inaddition, none of these labeling approaches determines the exact natureof the binding event, so for example active site binding to a receptoris indistinguishable from non-active-site binding such as allostericbinding, and thus no functional information is obtained via the presentdetection methodologies. Therefore, a method to detect binding eventsthat both eliminates the need for the label as well as yields functionalinformation would greatly improve upon the above mentioned approaches.

[0009] Other approaches for studying biochemical systems have usedvarious types of dielectric measurements to characterize certain classesof biological systems such as tissue samples and cellular systems. Inthe 1950's, experiments were conducted to measure the dielectricproperties of biological tissues using standard techniques for themeasurement of dielectric properties of materials known at the time.Since then various approaches to carrying out these measurements haveincluded frequency domain measurements, and time domain techniques suchas Time Domain Dielectric Spectroscopy. In these approaches, theexperiments were commonly carried out using various types of coaxialtransmission lines, or other transmission lines and structures oftypical use in dielectric characterization of materials. This includedstudies to look at the use and relevance of the dielectric properties ofa broad range of biological systems: The interest has ranged from wholetissue samples taken from various organs of mammalian species, tocellular and sub-cellular systems including cell membrane and organelleeffects. Most recently, there have been attempts to miniaturize theabove-mentioned techniques (see e.g., U.S. Pat. Nos. 5,653,939;5,627,322 and 5,846,708) for improved detection of changes in thedielectric properties of molecular systems. These configurations haveseveral drawbacks, including some substantial limitations on thefrequencies useable in the detection strategy, and a profound limitationon the sensitivity of detecting molecular systems, as well as beingexpensive to manufacture.

[0010] In general, limitations exist in the areas of specificity andsensitivity of most assay systems. Cellular debris and non-specificbinding often cause the assay to be noisy, and make it difficult orimpossible to extract useful information. As mentioned above, somesystems are too complicated to allow the attachment of labels to allanalytes of interest, or to allow an accurate optical measurement to beperformed. Further, a mentioned above, most of these detectiontechnologies yield no information on the functional nature of thebinding event. Therefore, a practical and economical universal enablingwhich can directly monitor without a label, in real time, the presenceof analytes or the extent, function and type of binding events and otherinteractions that are actually taking place in a given system wouldrepresent a significant breakthrough.

[0011] More specifically, the biomedical industry needs an improvedgeneral platform technology which has very broad applicability to avariety of water-based or other fluid-based physiological systems, suchas nucleic acid binding, protein-protein interactions, small moleculebinding, as well as other compounds of interest. Ideally, the assayshould not require highly specific probes, such as specific antibodiesand exactly complementary nucleic acid probes; it should be able to workin native environments such as whole blood, cytosolic mixtures, as wellas other naturally occurring systems; it should operate by measuring thenative properties of the molecules, and not require additional labels ortracers to actually monitor the binding event; for some uses it shouldbe able to provide certain desired information on the nature of thebinding event, such as whether or not a given compound acts as anagonist or an antagonist on a particular drug receptor, and not functionsimply as a marker to indicate whether or not the binding event hastaken place. For many applications, it should be highly miniaturizableand highly parallel, so that complex biochemical pathways can be mappedout, or extremely small and numerous quantities of combinatorialcompounds can be used in drug screening protocols. In many applications,it should further be able to monitor in real time a complex series ofreactions, so that accurate kinetics and affinity information can beobtained almost immediately. Perhaps most importantly, for mostcommercial applications it should be inexpensive and easy to use, withfew sample preparation steps, affordable electronics and disposablecomponents, such as surface chips for bio-assays that can be used for anassay and then thrown away, and be highly adaptable to a wide range ofassay applications.

[0012] It is important to note that other industries have similarrequirements for detection, identification or additional analysis. Whilemost applications involve the use of biological molecules, virtually anymolecule can be detected if a specific binding partner is available orif the molecule itself can attach to the surface as described below.

[0013] The present invention fulfills many of the needs discussed aboveand other needs as well.

SUMMARY OF THE INVENTION

[0014] The present invention provides test systems and bio-assay deviceswhich can be used to detect and identify molecular binding events. Inone embodiment, the invention provides a test system having a testfixture, a measurement system, and a computer. The test fixture includesa bio-assay device having a signal path and a retaining structureconfigured to place a sample containing molecular structures inelectromagnetic communication with the signal path. The measurementsystem is configured to transmit test signals to and to receive testsignals from the signal path at one or more predefined frequencies. Thecomputer is configured to control the transmission and reception of thetest signals to and from the measurement system.

[0015] The invention will be better understood when considered in lightof the foregoing drawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 illustrates one embodiment of a bio-assay system inaccordance with the present invention.

[0017]FIG. 2 illustrates one possible embodiment of a single path testsystem in accordance with the present invention.

[0018] FIGS. 3A-3F illustrate various views of a test fixture inaccordance with the present invention.

[0019]FIG. 4A illustrates a top view of a standard microstriptransmission line bio-assay for use with the test fixture of FIG. 3.

[0020]FIG. 4B illustrates a top view of a meandered transmission linebio-assay for use with the test fixture of FIG. 3.

[0021]FIG. 4C illustrates a top view of a ring resonator bio-assay foruse with the test fixture of FIG. 3.

[0022]FIG. 4D illustrates a top view of a capacitive gap bio-assay foruse with the test fixture of FIG. 3.

[0023]FIG. 4E illustrates a side view of a dielectric signal pathbio-assay for use with the test fixture of FIG. 3.

[0024]FIG. 5 illustrates one possible embodiment of an N×M array testsystem in accordance with the present invention.

[0025] FIGS. 6A-B illustrate various views of an N×M array test fixturein accordance with the present invention.

[0026]FIG. 7A illustrates one embodiment of a bio-assay array inaccordance with the present invention.

[0027]FIG. 7B illustrates one embodiment of an array element inaccordance with the present invention comprising a series-connected,electronically switched Field Effect Transistor.

[0028]FIG. 7C illustrates one embodiment of an array element inaccordance with the present invention comprising a series-connected,optically switched Field Effect Transistor.

[0029]FIG. 7D illustrates one embodiment of an array in accordance withthe present invention comprising two paths of two, serially-connectedFET devices.

[0030]FIG. 7E illustrates the circuit equivalent model of the arrayshown in FIG. 7D in accordance with the present invention.

[0031]FIG. 7F illustrates one embodiment of a two-dimensional bio-assayarray in accordance with the present invention.

[0032]FIG. 8 is an example of the effects of a protein bindingnon-specifically to the dielectric signal path of the bio-assay deviceillustrated in FIG. 4E.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0033] Table of Contents

[0034] I. Definitions

[0035] II. General Overview

[0036] III. Single Path Test System and Bio-Assays

[0037] A. Test System

[0038] B. Test Fixture

[0039] C. Bio-Assay Devices

[0040] IV. Array Test System and Bio-Assays

[0041] A. Test System

[0042] B. Test Fixture

[0043] C. Bio-Assay Devices

[0044] V. Applications

[0045] A. Drug Discovery Application

[0046] B. Nucleic Acid Chemistry Application

[0047] I. Definition of Terms

[0048] As used herein, the terms biological “binding partners” or“ligand/antiligand” or “ligand/antiligand complex” refers to moleculesthat specifically recognize other molecules to form proximal complexessuch as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleicacid, protein-protein, protein-small molecule such as drug-receptor,etc. Biological binding partners need not be limited to pairs of singlemolecules. Thus, for example, a single ligand may be bound by thecoordinated action of two or more “anti-ligands”.

[0049] As used herein, the term “ligand” or “analyte” or “marker” refersto any molecule being detected. It is detected through its interactionwith an antiligand, which specifically or non-specifically binds theligand, or by the ligand's characteristic dielectric properties. Theligand is generally defined as any molecule for which there existsanother molecule (i.e. an antiligand) which specifically ornon-specifically binds to said ligand, owing to recognition, chemical orotherwise, of some portion of said ligand. The antiligand, for example,can be an antibody and the ligand a molecule such as an antigen whichbinds specifically to the antibody. In the event that the antigen isbound to the surface and the antibody is the molecule being detected,for the purposes of this document the antibody becomes the ligand andthe antigen is the antiligand. The ligand may also consist of nucleicacids, proteins, lipids, small molecules, membranes, carbohydrates,polymers, cells, cell membranes, organelles and synthetic analoguesthereof.

[0050] Suitable ligands for practice of this invention include, but arenot limited to antibodies (forming an antibody/epitope complex),antigens, nucleic acids (e.g. natural or synthetic DNA, RNA, gDNA, cDNA,mRNA, tRNA, etc.), lectins, sugars (e.g. forming a lectin/sugarcomplex), glycoproteins, receptors and their cognate ligand (e.g. growthfactors and their associated receptors, cytokines and their associatedreceptors, signaling receptors, etc.), small molecules such as drugcandidates (either from natural products or synthetic analoguesdeveloped and stored in combinatorial libraries), metabolites, drugs ofabuse and their metabolic by-products, co-factors such as vitamins andother naturally occurring and synthetic compounds, oxygen and othergases found in physiologic fluids, cells, cellular constituents cellmembranes and associated structures, other natural products found inplant and animal sources, other partially or completely syntheticproducts, and the like.

[0051] As used herein, the term “antiligand” refers to a molecule whichspecifically or nonspecifically binds another molecule (i.e., a ligand).The antiligand is also detected through its interaction with a ligand towhich it specifically binds or by its own characteristic dielectricproperties. As used herein, the antiligand is usually immobilized on thesurface, either alone or as a member of a binding pair that isimmobilized on the surface. In some embodiments, the antiligand mayconsist of the molecules on the signal path, on a dielectric surface orin a dielectric volume, or a conductive surface. The antiligand mayfurther be attached by one or more linkers to a surface or matrixproximal to, or incorporated in, the signal path. Alternatively, once anantiligand has bound to a ligand, the resulting antiligand/ligandcomplex can be considered an antiligand for the purposes of subsequentbinding or other subsequent interactions.

[0052] As used herein, the term “specifically binds” when referring to aprotein or polypeptide, nucleic acid, or receptor or other bindingpartners described herein, refers to a binding reaction which isdeterminative of the cognate ligand of interest in a heterogeneouspopulation of proteins and/or other biologics. Thus, under designatedconditions (e.g. immunoassay conditions in the case of an antibody, orstringent conditions in the case of nucleic acid binding), the specifiedligand binds to its particular “target” (e.g. a hormone specificallybinds to its receptor, or a given nucleic acid sequence binds to itscomplementary sequence) and does not bind in a significant amount toother molecules present in the sample or to other molecules to which theligand or antibody may come in contact in an organism or in a samplederived from an organism.

[0053] As used herein, the terms “isolated” “purified” or “biologicallypure” refer to material which is substantially or essentially free fromcomponents that normally accompany it as found in its native state.

[0054] As used herein, the term “nucleic acid” refers to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogs of natural nucleotides that can function in a similar manner asnaturally occurring nucleotides.

[0055] As used herein, the terms “polypeptide”, “peptide” and “protein”are used interchangeably to refer to a monomer or polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers.

[0056] As used herein, the term “antibody” refers to a proteinconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes or fragments of immunoglobulin genes. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

[0057] A typical immunoglobulin (antibody) structural unit is known tocomprise a tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

[0058] Antibodies exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′) 2 dimer intoan Fab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include single chainantibodies, more preferably single chain Fv (scFv) antibodies in which avariable heavy and a variable light chain are joined together (directlyor through a peptide linker) to form a continuous polypeptide.

[0059] A single chain Fv (“scFv” or “scFv”) polypeptide is a covalentlylinked VH:VL heterodimer which may be expressed from a nucleic acidincluding VH- and VL-encoding sequences either joined directly or joinedby a peptide-encoding linker. Huston, et al. (1988) Proc. Nat. Acad.Sci. USA, 85:5879-5883. A number of structures for converting thenaturally aggregated—but chemically separated light and heavypolypeptide chains from an antibody V region into an scFv molecule whichwill fold into a three dimensional structure substantially similar tothe structure of an antigen-binding site. See, e.g. U.S. Pat. Nos.5,091,513 and 5,132,405 and 4,956,778.

[0060] An “antigen-binding site” or “binding portion” refers to the partof an immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complementaritydetermining regions” or “CDRs” and are characterized, for example byKabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987).

[0061] As used herein, the terms “immunological binding” and“immunological binding properties” refer to the non-covalentinteractions of the type which occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. As used herein,a biological sample is a sample of biological tissue or fluid that, in ahealthy and/or pathological state, that is to be assayed for theanalyte(s) of interest. Such samples include, but are not limited to,sputum, amniotic fluid, blood, blood cells (e.g., white cells), tissueor fine needle biopsy samples, urine, peritoneal fluid, and pleuralfluid, or cells therefrom. Biological samples may also include sectionsof tissues such as frozen sections taken for histological purposes.Although the sample is typically taken from a human patient, the assayscan be used to detect the analyte(s) of interest in samples from anymammal, such as dogs, cats, sheep, cattle, and pigs. The sample may bepretreated as necessary by dilution in an appropriate buffer solution orconcentrated, if desired. Any of a number of standard aqueous buffersolutions, employing one of a variety of buffers, such as phosphate,Tris, or the like, preferably at physiological pH can be used.

[0062] As used herein, the term “receptor” or “drug receptor” refers toa biological structure that is a target for drug therapy, and includesproteins such as membrane-bound structures like G-protein CoupledReceptors, nuclear receptors like hormone receptors; proteins whichmodulate the expression of genes, such as promoters and inducers;nucleic acid targets such as genes, expressed sequences, regulatory andsignaling sequences; other proteins in biological systems which modulateor mediate physiological activities of a given organism.

[0063] As used herein, the term “signal path” refers to a transmissionmedium along or through the bio-electrical interface which is capable ofsupporting an electromagnetic signal of any useful frequency including aDC static field. A non-exhaustive list of signal paths includeconductive and dielectric waveguide structures, conductive anddielectric transmission line structures, multiple-conductor and multipledielectric transmission mediums such as transverse electromagnetic (TEM)transmission lines, transmission lines with three or more conductive ordielectric elements which support Transverse Electric (TE), TransverseMagnetic (TM), or TEM modes of propagation such as quadrupolar andoctupolar lines; coupled waveguides and conductive and dielectricresonant cavity structures which may or may not be coupled; conductiveand dielectric antenna structures such as dipole and quadrupoleantennas; evanescent wave structures such as evanescent waveguides, bothcoupled and uncoupled, evanescent wave transmission lines, andevanescent wave antennas; other non-modal structures like wires, printedcircuits, and other distributed circuit and lumped impedance conductivestructures, and the like. In embodiments in which the signal pathconsists of a conductive region or regions, the conductive regionextends continuously over that range. In embodiments in which the signalpath is non-metallic, e.g., a dielectric waveguide, antenna, ortransmission line, the signal path is defined as the path having eitherthe greatest conductivity at the frequency or range of frequencies beingused, or as the molecular binding region itself.

[0064] As used herein, the term “molecular binding region” or “MBR”refers to a surface layer or a volume element having of at least onemolecular structure (i.e., an analyte, antiligand, or aligand/antiligand pair, etc.) coupled to the signal path along orbetween the bio-electrical interface. The molecular binding region mayconsist of one or more ligands, antiligands, ligand/antiligandcomplexes, linkers, matrices of polymers and other materials, or othermolecular structures described herein. Further, the molecular bindingregion may be extremely diverse and may include one or more componentsincluding matrix layers and/or insulating layers, which may have one ormore linking groups. The molecular binding region is coupled to thesignal path either via a direct or indirect physical connection or viaelectromagnetic coupling when the ligand is physically separated fromthe signal path. The molecular binding region may be of a derivatizedsurface such as by thiol linkers, alkanethiols, heterobifunctionalalkanes, branched dextrans, biotinylated metals and the like, all inaccordance with standard practice in the art.

[0065] As used herein, the term “binding event” refers to an interactionor association between two or more molecular structures, such as aligand and an antiligand. The interaction may occur when the twomolecular structures as are in direct or indirect physical contact orwhen the two structures are physically separated but electromagneticallycoupled. Examples of binding events of interest in a biological contextinclude, but are not limited to, ligand/receptor, antigen/antibody,drug-receptor, protein-protein, enzyme/substrate, DNA/DNA, DNA/RNA,RNA/RNA, nucleic acid mismatches, complementary nucleic acids andnucleic acid/proteins. Alternatively, the term “binding event” may referto a single molecule or molecular structure described herein, such as aligand, or an antiligand/ligand complex, which is bound to the signalpath. In this case the signal path is the second molecular structure.

[0066] As used herein, the term “ligand/antiligand complex” refers tothe ligand bound to the antiligand. The binding may be specific ornon-specific, and the bonds are typically covalent bonds, hydrogenbonds, immunological binding, Van der Waals forces, or other types ofbinding.

[0067] As used herein, the term “coupling” refers to the transfer ofenergy between two structures either through a direct or indirectphysical connection or through any form of signal coupling, such aselectrostatic or electromagnetic coupling, matter-field interactions,and the like.

[0068] As used herein, the term “test signal” refers to a d.c, frequencydomain, or time domain signal used to probe the bio-assay device.Frequency domain signals may propagate at any useful frequency definedwithin the electromagnetic spectrum. For example, the frequency rangewithin which a test signal may propagate is for example at or above 1MHz, such as 5 MHz 10 MHz, 20 MHz, 45 MHz, 100 MHz, 500 MHz, 1 GHz, 5GHz, 10 GHz, 30 GHz, 50 GHz, 100 GHz, 500 GHz, 1000 GHz and frequenciesranging therebetween. Time domain test signals may be generated insquare, sawtooth, triangle, or other known waveforms and propagate atperiodic or aperiodic intervals, Time domain signals may consist ofamplitudes and rise/fall times which permit modulation which coupled tothe molecular binding region. For example, a time domain test signal mayconsist of a square waveform having an amplitude between 0V and 50V, anda rise/fall time of between 0.1 pS and 1 uS, or range anywheretherebetween.

[0069] As used herein, the term “enzyme,” refers to a protein which actsas a catalyst to reduce the activation energy of a chemical reaction inother compounds or “substrates”, but is not a final product in thereaction.

[0070] As used herein, the term “sample” and/or “solution” includes amaterial in which a ligand resides. A non-exhaustive list of solutionsincludes materials in solid, liquid or gaseous states. Solid solutionsmay be comprised of naturally-occurring or synthetic molecules includingcarbohydrates, proteins, oligonucleotides, or alternatively, any organicpolymeric material, such as nylon, rayon, dacryon, polypropylene,teflon, neoprene, delrin or the like. Liquid solutions include thosecontaining an aqueous, organic or other primary components, gels, gases,and emulsions. Exemplary solutions include celluloses, dextranderivatives, aqueous solution of d-PBS, Tris buffers, deionized water,blood, physiological buffer, cerebrospinal fluid, urine, saliva, water,organic solvents. The solution is used herein to refer to the materialin which the ligand and/or antiligand are applied to the bindingsurface. The solution contains the sample to be analyzed.

[0071] As used herein, the term “linking group” or “linker” refers tochemical structures which are used to attach any two components on thebio-assay device. The linking groups thus have a first binding portionthat binds to one component, such as a conductive surface or dielectricmatrix, and have a second binding portion that binds to anothercomponent such as the matrix or the antiligand.

[0072] As used herein, the term “bio-assay device” refers to a structureon which the molecular binding region is formed. The bio-assay devicemay consist of a surface, recessed area, volume, or a hermeticallysealed enclosure, each of which may be any particular size or shape.

[0073] As used herein, the “bio-assay system” refers to the bio-assaydevice as described above, in connection with the components necessaryto electromagnetically probe and detect the bio-assay device. Thesecomponents include, but are not limited to, the signal path(s),substrate(s), electronic devices such as signal generators,oscilloscopes, network analyzers, time domain reflectometers or otherequipment necessary to probe and detect signals from the bio-assaydevice, microchips and microprocessors which can probe and detectelectromagnetic signals and analyze data, and the like.

[0074] As used herein, the term “resonant” or “resonance” refersgenerally to a rapidly changing dielectric response as a function offrequency.

[0075] As used herein, the term “dispersion” refers to the functionaldependence of the dielectric properties of a material on the frequencyof the probing radiation, and in particular is used to distinguishregions of the electromagnetic spectrum in which the dielectricproperties of a given material has a strong functional dependence on thefrequency of the probing electromagnetic energy.

[0076] As used herein, “bio-electrical interface” refers to an interfaceregion which includes the signal path for supporting test signalpropagation and the molecular binding region of a sample.

[0077] As used herein, the term “matrix” or “binding matrix” refers to alayer or volume of material on the bio-assay chip that is used as aspacer or to enhance surface area or volume available for binding or tooptimize orientation of molecules for enhanced binding, or to enhanceany other property of binding so as to optimize the bio-assay device.The matrix layer may be comprised or carbohydrates such as dextran, polyamino acids, cross-linked and non-cross linked proteins, and the like.

[0078] As used herein, the term “structural change” refers to any changeof position, chemical make-up, orientation, conformation, relativeorientation of sub-structures or sub-units of a molecule or molecularsystem. A non-exhaustive list includes conformational changes,dimerization and polymerization, covalent binding, sub-unit motion,interactions with other molecules such as covalent and non-covalentbinding, hydrophobic bonding, denaturation and re-naturation,hybridization, ionization, substitution, and the like.

[0079] II. General Overview of the Bio-Assay System

[0080] The present invention makes use of the observation that a vastnumber of molecules can be distinguished based upon the uniquedielectric properties most molecules exhibit. These distinguishingdielectric properties can be observed by coupling an electromagneticsignal to the bound molecular structure. The unique dielectricproperties modulate the signal, giving it a unique signal response. Theunique signal response can then be used to detect and identify theligands and other molecules which make up the molecular binding region.

[0081]FIG. 1A illustrates a side view of one embodiment of a bio-assaysystem 100 in accordance with the present invention. The system 100 isillustrated in a two conductor, signal-plane ground-plane, circuittopology which may be realized in a multitude of architectures includinglumped or distributed element circuits in microstrip, stripline,coplanar waveguide, slotline or coaxial systems. Moreover, those ofskill in the art of electronics will readily appreciate that the systemmay be easily modified to a single conductor waveguide system, or athree or more conductor system.

[0082] As illustrated, the system 100 includes a signal source 110,transmission lines 120, a ground plane 130, a bio-assay device 150, anda signal detector 160. The illustrated embodiment shows two transmissionlines 120 coupled to the bio-assay device 150, although in analternative embodiment, the system may consist of a single transmissionline coupled to the bio-assay device for making a single portmeasurement. Further alternatively, three or more transmission lines maybe coupled to the bio-assay device 150 for multiple port measurements.

[0083] Transmission lines 120 are formed from a material which cansupport the propagation of a D.C voltage/current of an A.C. time orfrequency domain signal over the desired frequency of operation.Transmission lines 120 may be realized as a conductive layer, such as acenter conductor in a coaxial cable or a gold transmission line,deposited on a substrate, such as alumina, diamond, sapphire, polyimide,or glass using conventional photolithography or semiconductor processingtechniques. Signal interconnections 122 may be made via wire/ribbonbonds, soldering, conductive epoxy, connectors, or other conventionalconnection techniques appropriate for the frequency of operation.

[0084] The system 100 further includes a bio-assay device 150 whichincludes a dielectric substrate 151 and a signal path 152. Thedielectric substrate 151 may consists of any insulating material such asglass, alumina, diamond, sapphire, silicon, gallium arsenide orinsulating materials used in semiconductor processing. Alternatively,dielectric material such as RT/Duroid® manufactured by the RodgersCorporation or other similar dielectric materials may be used.

[0085] The signal path 152 is designed to provide a low insertion lossmedium and can consist of any TE, TM, or TEM signal architecture. In anexemplary embodiment, the signal path 152 consists of aphotolithographically formed microstrip transmission line having asputtered gold thickness on the order of between 0.1 um to 1000 um. Inthis embodiment, the transmission line is designed to provide low signalloss from D.C. to 110 GHz. Other condutive materials such as indium tinoxide (ITO), copper, silver, zinc, tin, antimony, gallium, cadmium,chromium, manganese, cobalt, iridium, platinum, mercury, titanium,aluminum, lead, iron, tungsten, nickel, tantalum, rhenium, osmium,thallium or alloys thereof may be used to form the transmission line. Inanother embodiment, the signal path 152 consists a dielectric region,further described below.

[0086] A bio-electrical interface region 153 defines the region over thesignal path 152 and the MBR 156 of the applied sample 157 areelectromagnetically coupled. In one embodiment of the invention, the MBR156 specifically binds to the signal path 152. In another embodiment ofthe invention, the MBR 156 binds non-specifically to the signal path152. In still another embodiment of the invention, the MBR iselectromagnetically coupled to, but is separate from the signal path152. Sufficient electromagnetic coupling may occur either through directbinding to the signal path 152 or from the molecular structures of theMBR 156 being suspended in close proximity to the signal path 152. Whendirect molecular binding to the signal path is sought, the signal pathmay include linker and/or matrix layers as further described in thecommonly-owned, co-pending U.S. patent application entitled “Method andApparatus for Detecting Molecular Binding Events,” Ser. No. 09/243,194,filed Feb. 2, 1999 incorporated herein by reference.

[0087] The MBR 156 is primarily composed of one or more ligands,although other molecules and structures may also be included, asdescribed herein. The MBR 156 may consist of only one bound ligand tier,for instance in the case of primary binding, or it may consist of two,three, four, five or more bound ligand tiers, in the instances wherethere are secondary or higher-order binding events occurring. Multipleligand tiers may occur at different binding surfaces 155 over the samesignal path. Additionally, the MBR 156 may comprise a matrix in avolume, with ligands and antiligands attached to structural componentssuch as branched dextran, polymers, amino acid chains, other linkersknown in the art, and the like.

[0088] In the illustrated embodiment, dielectric substrate 151 islocated between the signal path 151 and the ground plane 159. However,the MBR 156 and sample 157 may be located proximate to the ground plane159 such that MBR 156 is electromagnetically coupled to ground plane 159alternatively or in addition to the MBR's location to the signal path152 as shown in FIG. 1A.

[0089] The system 100 includes a signal source 110 which launches a testsignal 112 onto the transmission line 120 and towards the bio-assaydevice 150. A signal detector 160 is positioned along the transmissionpath to receive the modulated test signal 162 (either reflected ortransmitted or both). When the test signal 120 propagates along thebio-electrical interface region 153 of the bio-assay device 150, thedielectric properties of the MBR 156 modulate the test signal. Themodulated test signal 162 is then recovered by the detector 160 and usedto detect and identify the molecular binding events occurring within theMBR 156.

[0090]FIG. 1B illustrates a second embodiment of the bio-assay testsystem in accordance with the present invention. Reference numbers usedin FIG. 1A are reused to indicate previously described elements. Thesystem includes the described signal source 110, transmission lines 120,connections 122, ground plane 130, bio-assay device 150 and signaldetector 160.

[0091] The bio-assay device 170 includes a dielectric substrate 151 andground plane 159, previously described. The signal path includestransmission lines 172 and a dielectric region 156 formed across thebio-electrical interface region 153 between transmission lines 120. Thedielectric region 156 is composed of the MBR and formed from themolecular binding events of the sample 157. The dielectric region isdesigned to provide a DC-blocked, low signal loss medium betweentransmission lines 172. The D.C. blocking properties of the dielectricregion 156 prevents D.C. voltages and currents from passing between theinput and output which could interfere with the operation of the testsystem, further described below. Dielectric region 156 provides lowsignal loss over the desired testing frequencies, some examples being 1MHz, 5 MHz 10 MHz, 20 MHz, 45 MHz, 80 MHz, 100 MHz, 250 MHz, 500 MHz,750 MHz, 1 GHz, 2.5 GHz, 5 GHz, 7.5 GHz, 10 GHz, 12 GHz, 18 GHz, 20 GHz,22 GHz, 24 GHz, 26 GHz, 30 GHz, 33 GHz, 40 GHz, 44 GHz, 50 GHz, 80 GHz,96 GHz, 100 GHz, 500 GHz, 1000 GHz, or frequencies ranging therebetween.

[0092] As described above, the MBR operates to modulate the test signal.The architecture of the dielectric region 156 serves to signal supportpropagation through the bio-electrical interface region without highsignal loss. An insulating substrate 176 is used as a binding surfacefor the MBR in order to form the dielectric region 156 and the MBR maybind either specifically or non-specifically to the insulating substrate176. The insulating substrate 151 may consist of the same or differentdielectric material as the dielectric substrate 151 and may,alternatively or in addition, consist of linker, matrix, and/orinsulating layers further described in the incorporated patentapplication entitled: “Method and Apparatus for Detecting MolecularBinding Events,” Ser. No. 09/243,194.

[0093] The length of the dielectric region (MBR) 156 is selected toprovide sufficient test signal modulation while minimizing through loss.Typical lengths are on the order of 10⁻¹m, 10⁻²m 10⁻³m, 10⁻⁴m, 10⁻⁵m,10⁻⁶m, 10⁻⁷m, 10⁻⁸m, 10⁻⁹m, 10⁻¹⁰m 10⁻¹¹m, or range anywheretherebetween.

[0094] As indicated, detection and identification of a ligand is alsopossible when the ligand is physically separated from butelectromagnetically coupled to the signal path 151. In this instance,the coupling between the signal path 151 and the suspended ligand willalter the response of the test signal propagating along the signal path151, thereby providing a means for detecting and/or identifying it. Themaximum separation between the signal path 151 and suspended ligand isinfluenced by such factors as the effective dielectric constant of themedium between the signal path 151 and the ligand, the total couplingarea, the sensitivity of the signal detector, concentration of theligands in solution, and the desired detection time. Separationdistances are typically on the order of 10⁻¹m, 10⁻²m 10⁻³m, 10⁻⁴m,10⁻⁵m, 10⁻⁶m, 10⁻⁷m, 10⁻⁸m, 10⁻⁹m, 10⁻¹⁰m or range anywheretherebetween.

[0095] In some embodiments, such as cell based assays, the MBR 156 maybe electromagnetically coupled to the signal path 151 through thesample. Thus, cells, and in particular cell membranes and membrane-basedstructures may couple to the signal path indirectly.

[0096] III. Single Path Test System and Bio-assay

[0097] Molecular binding events occurring within the MBR maybe detectedand identified using various test systems which generate, recover, andsubsequently analyze changes in the generated test signal. Test systemswhich are capable of use with the present invention include thosesystems designed to detect changes in the signal's voltage, current,impedance, admittance, reactance, amplitude, phase, delay, frequency,wave shape and/or timing, and other signal properties.

[0098] A. Test System

[0099]FIG. 2 illustrates one possible embodiment of a single path testsystem 200 in accordance with the present invention. The test systemincludes a test fixture 300, further described below, a measurementsystem 240 and a computer 260. Measurement system 240 communicates testsignals to and from test fixture 300 via test cables 224. Computer 260controls measurement system 240 via a control bus 250.

[0100] In one embodiment, measurement system 240 includes an S-ParameterTest Module model no. 8516A, a Frequency Synthesizer (not shown) modelno. 8341B, and a Vector Network Analyzer model no. 8510B, all of whichare manufactured by the Hewlett Packard Company of Palo Alto, Calif.(www.hp.com). In this embodiment, measurement system 240 provides ameasurement capability between the frequencies of 45 MHz and 40 GHz. Inan alternative embodiment, measurement system 240 may consist of modelnumber HP 8751A network analyzer which provides a measurement capabilitybetween 5 Hz and 500 MHz. In a further embodiment, measurement systemmay consist of model number HP 85106D which provides a measurementcapability between 33 GHz and 110 GHz, both manufactured by the HewlettPackard Company. Other measurement systems such as scalar networkanalyzers, Time Domain Reflectometers, an other similar measurementsystems may also be used to detect a change in the test signal which isattributable to the dielectric properties of the MBR.

[0101] Test cables 224 support the propagation of the test signals atthe desired frequency. In one embodiment, test cables consists of modelnumber 6Z PhaseFlex™ Microwave test cables manufactured by the W. L.Gore and Associates, Inc. of Newark Del. (www.gore.com). Control bus 250provides communication between the test system and computer 260 and inthe illustrated embodiment consists of a General Purpose Instrument Bus(GPIB). In alternative embodiments, measurement system 240 and computer260 may be integrated within a single automated measurement unit.

[0102] Computer 260 controls measurement system 240 to generate testsignals at one or more frequencies, output power levels, signal shapes,phase offsets or other measurement settings. In the preferredembodiment, computer 260 includes a +450 MHz microprocessor, such asthose manufactured by the Intel Corporation of Santa Clara, Calif.(www.intel.com). Test system control, data acquisition, and analysis maybe performed using a graphical programming software tool, such asLabVIEW® manufactured by the National Instruments Corporation of Austin,Tex. (www.natinst.com).

[0103] Alternatively or in addition, measurement system 240 may includea Time Domain Reflectometer (TDR) system, such as those optionallyavailable with the above-described network analyzers or described in theincorporated patent application entitled: “Method and Apparatus forDetecting Molecular Binding Events,” Ser. No. 09/243,194. Essentially,TDR systems transmit a signal pulse towards a unit under test. Thereturn signal (either reflected from or transmitted through the unitunder test) can be analyzed to ascertain information about the unitunder test. Specifically in the present embodiment, the dielectricproperties of the MBR will modulate the signal pulse, thereby enablingdetection and identification of the molecular binding events therein.

[0104] TDR measurements may be made at the fixture level using theaforementioned systems, or at the bio-assay device level utilizing oneor more of the standard techniques of microwave monolithic circuit(MMIC) technologies. When a TDR measurement is made at the device level,a time-domain test signal is generated in close proximity to thebio-assay device. This signal is then propagated along the signal pathto the bio-assay element via standard conductive geometries used in MMICtechnologies. The molecular binding region modulates the time-domaintest signal, and the modulated signal is then recovered to be analyzed.

[0105] B. Test Fixture

[0106] The test fixture of the present invention is designed to providea signal path and to secure the MBR of the applied sample in directcontact with or in close proximity to the signal path such that a testsignal propagating therealong will electromagnetically couple to theMBR. The test fixture may consist of a wholely or partially enclosed, orrecessed structure over or into which the sample may be deposited,injected, or otherwise applied.

[0107]FIG. 3A illustrates in a side view one possible embodiment of thetest fixture 300 in accordance with the present invention. Test fixture300 includes a top plate 302 and a bottom plate 304. Top plate 302includes ports 350 a and 350 b for injecting the sample solution. Topplate 302 further includes the top half of a sample cavity 340 a. Bottomplate 304 includes the bottom half of the sample cavity 340 b. In thepreferred embodiment, top and bottom plates 302 and 304 are eachcomposed of machined stainless steel and each measures 0.0320 cm×1.575cm×3.15 cm.

[0108] Contained with the sample cavity 340 is a reaction vessel 310, anO-ring 320, a bio-assay device 400 (further described in FIG. 4 below),and a bottom spacer 330. Reaction vessel 310 includes ports 312 a and312 b for receiving the sample. Reaction vessel 310 further includes anO-ring cavity 318 for accommodating the O-ring 320. O-ring 320 ispositioned between the reaction vessel 310 and the bio-assay device 400to secure the sample along the bio-assay device 400. Bio-assay device400 provides the signal path and bioelectrical interface along which theMBR will form. Bottom spacer 330 is provided to elevate the bio-assaydevice 400 to the proper height so that it may couple to input andoutput transmission lines (not shown) formed between the top and bottomplates 302 and 304.

[0109] The sample is injected into sample cavity 340 via feed tubes (notshown) coupled to ports 350 a and 350 b. Sample flows through reactionvessel ports 312 a and 312 b into the reaction vessel 310. In thepreferred embodiment, the sample is injected by applying positivepressure in one feed tube and negative pressure to the other feed tube.

[0110]FIG. 3B illustrates an end view of the test fixture shown in FIG.3A. As illustrated, test fixture 300 includes connectors 360 a and 360 bfor communicating signals into and/or out of the test fixture 300.Connectors 360 a and 360 b are secured to top and bottom plates 302 and304 via screws 361. Connectors 360 and 360 include center conductors 362which are coupled to the bio-assay device 400 via transmission lines(not shown) formed between the top and bottom plates 302 and 304,respectively. In the preferred embodiment, connectors 360 are SMAconnectors such as those manufactured by the SRI Connector Gage Companyof Melbourne, Fla. (www.sriconnectorgage.com). In alternativeembodiments, connectors 360 may consist of N, 3.5 mm, 2.9 mm, 2.4 mm orother connectors appropriate for the test frequency range.

[0111]FIG. 3C illustrates a top view of top plate 302 showing ports 350a and 350 b and top half of sample cavity 340 a. In its preferredembodiment, top half of sample cavity 340 a measures 0.4 cm×0.4 cm×0.080cm. FIG. 3D illustrates a top view of bottom plate 304 showing thebottom half of sample cavity 340 b, also measuring 0.40 cm×0.40 cm×0.080cm in the preferred embodiment. FIGS. 3E and 3F illustrate side andbottom views respectively of reaction vessel 310. In its preferredembodiment, reaction vessel is composed of Lexan® and measures 0.4cm×0.4 cm×0.070 cm. Ports 312 a and 312 b are 0.030 cm diameter. O-ringcavity 318 has an diameter of 0.240 cm.

[0112]FIGS. 3G and 3H illustrate top and side views of O-ring 320,respectively. In the preferred embodiment, O-ring 320 is composed of anelastomer, such as Viton® and measures 0.100 cm×0.240 cm with an innerdiameter of 0.030 cm. FIG. 3I and 3J illustrate top and side views ofbottom spacer 330. In the preferred embodiment, bottom spacer iscomposed of Lexan® or alumina and measures 0.4 cm×0.4 cm×0.025 cm.

[0113] C. Bio-Assay Device

[0114] The bio-assay device forms the bio-electrical interface of thepresent detection system. The device includes a signal pathelectromagnetically coupled to the MBR. One or more input/output portsare connected to the signal path to communicate the test signal. Asingle input/output port may be used, when for instance a reflectionmeasurement, known in the art, is sought. Alternatively, separate inputand output ports may be used when a through measurement, also known inthe art, is sought alternatively or in addition to the reflectionmeasurement.

[0115] The signal path is preferably formed along a direction which isnon-orthogonal to the MBR. In one embodiment, the test signal propagatesin parallel to a tangent on the surface on which the MBR is formed. Inother embodiments, the test signal may propagate at an angle of ±1°,±2°, ±3°, ±4°, ±5°, ±10°, ±15°, ±20°, ±30°, ±40°, ±45°, ±50°, ±60°,±70°, ±80°, or ±85° relative to the MBR binding surface, or any rangestherebetween. In a first embodiment, the signal path consists of atransmission line in a two conductor structure and the direction of thesignal path is defined by the Poynting vector as known in the art ofelectromagnetics. In a second embodiment, the transmission line mayconsist of a conductive region or layer which extends continuously alongthe bio-electrical interface region. In a third embodiment, the signalpath maybe defined as the path having the least amount of signal lossalong the bio-electrical interface over the desired frequency range ofoperation. In a fourth embodiment, the signal path maybe defined ashaving an a-c. conductivity of greater than 3 mhos/m, i.e., having aconductivity greater than that a saline solution, typically greater than5 mhos/m, but ideally in the range of 100 to 1000 mhos/m and greater. Asdescribed above, the MBR may be either be in direct contact with orphysically separated from but electromagnetically coupled to the signalpath.

[0116] The signal path may be realized in a number of differentarchitectures, such as a conductive wire, a transmission line, aconductive or dielectric waveguide structure, a resonant cavity, or anyother transmission medium that will support the propagation of the testsignal over the desired frequency range. At high test frequencies(frequencies above 10 MHz, for example) the signal path may be realizedin microstrip, stripline, suspended substrate, slotline, coplanarwaveguide, conductive or dielectric waveguide, or other high frequencysignal path architectures such as those described in R. E. CollinsFoundations for Microwave Engineering, McGraw-Hill Publishing Co., 1966;and S. March, Microwave Transmission Lines and Their PhysicalRealizations, Les Besser and Associates, Inc., 1986. The followingexamples are but a few of the possible signal path embodiments withinthe scope of the present invention.

[0117] Through Microstrip Transmission Line

[0118]FIG. 4A illustrates a top view of a standard microstriptransmission line bio-assay 410 for use with the test fixture of FIG.3A. As illustrated, the signal path consists of a transmission line 412of width of 0.065 cm and length of 1.0 cm between the input/output ports411. Bio-assay 410 is formed using standard photolithographic techniquesand fabricated using sputtered gold transmission lines on a 0.55 mmthick quartz glass substrate having a dielectric constant of approx. 3.Those of skill in the art will appreciate that other signal patharchitectures, conductive and substrate materials, and photolithographictechniques may be alternatively employed.

[0119] During a testing operation, a sample is applied over thetransmission line 412 and a MBR is formed along the exposed surface ofthe transmission line 412. The MBR may be either in direct physicalcontact with the transmission line 412 or separated from butelectromagnetically coupled to the line 412. In the embodiment in whichthe MBR is in direct contact with the transmission line, linker and/ormatrix layers may be employed to facilitate binding thereto as furtherdescribed in the incorporated patent application entitled: “Method andApparatus for Detecting Molecular Binding Events,” Ser. No. 09/243,194.

[0120] Next, a test signal is launched on to the transmission line 412through, for example, an SMA type connector 360, shown in FIG. 3B. Asthe test signal propagates along the transmission line portions have aMBR attached or in close proximity thereto, the dielectric properties ofthe MBR modulate the test signal. The modulated test signal is then berecovered and used to detect and identify the molecular binding eventsoccurring within the MBR.

[0121] Meandered Microstrip Transmission Line

[0122]FIG. 4B illustrates a top view of a meandered transmission linebio-assay 420 for use with the test fixture of FIG. 3A. Bio-assay 420includes a meandered line coupled between an input/output ports 421. Themeander line 422 is designed to increase the MBR surface area whichprovides greater measurement sensitivity, while adding minimal lengthand size to the detection structure.

[0123] In the illustrated embodiment, the meandered line 422 has a widthof 0.065 cm and length of 1.0 cm between the input/output ports 422.Transmission line corners may be mitered, 45° to minimize signalreflection and maximize signal transmission along the line 422. Spacing424 is designed to minimize coupling between proximate line sections. Inone embodiment, line spacing is 0.033 cm. In an alternative embodimentline spacing 424 is defined such that coupling between proximate linesections 422 a, 422 b is no more than −7 dB. Bio-assay 420 is formedusing standard photolithographic techniques and fabricated usingsputtered gold transmission lines on a 0.55 mm thick quartz glasssubstrate having a dielectric constant of approx. 3. Those of skill inthe art will appreciate that other signal path architectures, conductiveand substrate materials, and photolithographic techniques may bealternatively employed.

[0124] During a testing operation, a sample is applied over themeandered line 422 and a MBR is formed along the exposed surface of themeandered line 422. The MBR may be either in direct physical contactwith the meandered line 422 or separated from but electromagneticallycoupled to the line 422. Linker and/or matrix layers may be used tofacilitate binding to the meandered line 422.

[0125] Next, a test signal is launched on to the transmission line 422through, for example, an SMA type connector 360, shown in FIG. 3B. Asthe test signal propagates along the transmission line portions have aMBR attached or in close proximity thereto, the dielectric properties ofthe MBR modulate the test signal. The modulated test signal is then berecovered and used to detect and identify the molecular binding eventsoccurring within the MBR.

[0126] Numerous variations in the illustrated design may be realized toincrease the detection sensitivity over a minimum detection area. Forinstance, when employed miters may be designed to provide an intentionalimpedance mismatch between line segments, thereby causing signalreflections between miters. When the effective signal length of the linesegment approaches 180 degrees, the reflected signals will combine inphase with incoming signals, thereby a larger amplitude output signal atthese frequencies. Higher output power permits greater measurementsensitivity and the length of the line segments can be tune to detect ormore closely inspect responses occurring at specific frequencies.

[0127] Microstrip Ring Resonator

[0128]FIG. 4C illustrates a top view of a ring resonator bio-assay 430for use with the test fixture of FIG. 3A. The bio-assay 430 includesinput/output ports 431 a and 431 b coupled to a ring resonator 434. Ringresonator 434 includes three concentric rings 434 a-c and a solidcircular ring 434 d disposed therein. Each ring 434 a-c has a width of0.1 cm and is separated from proximate ring(s) by a spacing of 0.1 cm.The solid circular element 434 d is 0.050 cm in radius and is disposedat the ring center. In alternative embodiments, spacing 434 e and/orwidths may vary from ring to ring. Bio-assay 430 is formed usingstandard photolithographic techniques and fabricated using sputteredgold transmission lines on a 0.55 mm thick quartz glass substrate havinga dielectric constant of approx. 3. Those of skill in the art willappreciate that other signal path architectures, conductive andsubstrate materials, and photolithographic techniques may bealternatively employed.

[0129] During normal operation without an applied sample, a test signalis injected into the port 431 a through, for example, an SMA connector360 as shown in FIG. 3B. Via electromagnetic coupling, a portion of thetest signal propagates through the ring resonator 434 and to the outputport 431 b. An impedance mismatch occurs at this interface 431 b,reflecting a portion of the signal back toward the source interface 431a. The remaining portion of the signal propagates out of the resonantcircuit along the input line segment and to the test set. At the sourceinterface 431 a, a second impedance mismatch occurs and reflecting aportion of the reflected signal again toward the resonator output 431.The remaining portion of the signal is propagated out of the resonantcircuit along the output line segment toward the test set input. Thesignal continues to “ping-pong” between the interfaces 431 a and 431 buntil the signal is dissipated or transmitted to the source or test set.The magnitude of the reflected wave depends in part on the magnitude ofthe impedance mismatch at the interfaces 431 a and 431 b. The larger theimpedance mismatches, the larger the reflected signal.

[0130] At one or more frequencies, the effective signal path betweeninterfaces 431 a and 431 b approaches a 180° phase shift (or a multiplethereof). When this occurs, the reflected signal will reach inputinterface 431 a having a phase substantially equal to the phase of theincoming signal. In this instance, the incoming signal and the reflectedsignal will recombine in-phase, thereby producing a stronger signal.When the stronger signal reaches the output interface 431 b, a largermagnitude signal (compared to the non-combined signal) will exit fromthe output interface 431 b to the test set. Thus, the resonator 434 willoutput a larger magnitude signal near frequencies in which the resonator434 has an effective signal length near 180° or a multiple thereof. Thisdifference in output signal strength can be monitored and detected usingthe measurement systems described herein.

[0131] When the sample is applied over the resonator 430, a MBR isformed along the exposed portion of rings 434 a-d. The MBR may either bein direct physical contact with the rings or separated from butelectromagnetically coupled to the rings 434 a-d. Linker and/or matrixlayers may be employed to facilitate binding to the resonator rings 434a-d and/or input and output interfaces 431 a and 431 b.

[0132] Next, a test signal is injected into the input port 431 a asabove. The test signal couples between rings of the resonator 434 asbefore, except that the dielectric properties of the MBR operates tochange the frequency(s) at which the resonator 434 approaches 180°.Further, because the dielectric properties of each different MBR aredistinct, each MBR will produce a different “frequency marker”, i.e.,the frequency at which the resonator approaches a 180° phase shift andproduces a larger output signal. In this manner, samples containingdifferent molecular structures will exhibit different frequency markers,which can be used to detect their presence in an unknown solution. Inaddition, molecular structures within a particular class, alpha-helices,beta-sheets and other structural motifs in proteins may exhibit“related” frequency markers, e.g., frequency markers within closeproximity to each other or frequency markers which occur within apredictable pattern.

[0133] Those of skill in the art of Microwave engineering willunderstand that other resonant structures are also possible. Forinstance, the resonator 434 may alternatively consist of a transmissionline segment connected between the input and output interfaces 431 a and431 b. In this embodiment, the transmission line segment will have theappropriate impedance relative to the input and output ports to providethe desired input and output impedance mismatch and the appropriatelength to provide the 180° phase shift in presence of the sample. Otherresonant configurations such as a proximately placed dielectric puck aswell as others may be used with minor modifications to detect thepresence or absence of particular molecular structures.

[0134] Microstrip Capacitive Gap

[0135]FIG. 4D illustrates a top view of a capacitive gap bio-assay 440for use with the test fixture of FIG. 3A. Bio-assay 440 includes aninput port 441 a coupled to an input line segment 442 a and an outputport 441 b coupled to an output line segment 442 b. Disposed between theinput and output line segments 442 a and 442 b is a gap 444 where thesample is deposited during testing. In the illustrated embodiment, inputand output line segments 442 a and 442 b are each 0.495 mm long and0.250 mm wide. Capacitive gap 444 measures 0.010 mm×0.250 mm. Bio-assay440 is formed using standard photolithographic techniques and fabricatedusing sputtered gold transmission lines on a 0.55 mm thick quartz glasssubstrate having a dielectric constant of approx. 3. Those of skill inthe art will appreciate that other signal path architectures, conductiveand substrate materials, and photolithographic techniques may bealternatively employed.

[0136] During normal operation without an applied sample, a test signalis injected into the port 441 a through, for example, an SMA connector360 as shown in FIG. 3B. Via electromagnetic coupling, a portion of thetest signal's electromagnetic field propagates across the capacitive gap444 between the input and output line segments 442 a and 442 b. Thecapacitive gap 44 prevents the transmission of D.C. voltage and currentfrom passing between the input and outputs. The test signal is thenrecovered at the output port 441 b for processing. The width andseparation of the gap 444, impedances of input and output line segments442 a and 442 b, the dielectric constant of the substrate 445, and thefrequency of operation will influence the amount of signal powertransferred between the input and output ports 441 a and 441 b. Thecapacitive gap circuit 440 will exhibit a signal response which variesover a test frequency range.

[0137] When the sample is applied over the gap 444, a MBR is formedalong the edges of input and output line segments 442 a and 442 b. TheMBR may either be in direct physical contact with the line segment edges442 a and 442 b, or separated from but electromagnetically coupledthereto. Linker and/or matrix layers may be used on the line segments442 a and 442 b to promote molecular binding thereto.

[0138] The formation of the MBR on gap edges effects the signal'stransmissivity from the input port 441 a to the output port 441 b.Specifically, the MBR creates a gap circuit, the response of whichvaries over the test frequency range. As described above, each distinctMBR will exhibit a different dielectric property which serves to createa distinct frequency response or “signature.” The frequency signature ofa known molecular sample can stored and later used to identify themolecular structure in an unknown solution. Molecular structures withinthe same class may exhibit a similar frequency pattern over a commontest frequency range. In this case, the tester is able to identify theclass of the unknown molecular structure if the identity of themolecular structure itself is known.

[0139] The capacitive configuration may be used as a single detectionelement or in combination with one or more of the detection elementslisted herein to enhance, tune, or detune the frequency response at oneor more frequencies.

[0140] Dielectric Signal Path

[0141]FIG. 4E illustrates a side view of a dielectric signal pathbio-assay 450 having for use with the test fixture of FIG. 3. Bio-assay450 includes an input line segment 451, an output line segment 452formed on a dielectric substrate 456, and a dielectric region 455disposed between the input and output line segments 451 and 452. Thebottom surface of dielectric region 455 is formed by insulatingsubstrate 453 which is treated to promote molecular binding thereto. Theinsulating substrate 453 may consist of the same or different materialas the dielectric substrate 456. Further, the insulating substrate 453may include of linker and/or matrix layers, further described in thecommonly owned, copending U.S. patent application entitled “Method andApparatus for Detecting Molecular Binding Events,” Ser. No. 09/243,194,filed Feb. 2, 1999 incorporated herein by reference. In the exemplaryembodiment of FIG. 4E, the bio-assay 450 is fabricated using standardmicrostrip photolithographic techniques on a dielectric substrate 456 of0.55 mm quartz glass substrate having a dielectric constant ofapproximately 3. The dielectric region 455 is 100 Angstroms deep andextends 2.5 um between the input and output line segments 451 and 452.

[0142] When a sample 456 is applied over the dielectric region 455, alongitudinal MBR 457 is formed along the surface of the insulatingsubstrate 453. The formed MBR serves as a signal path for the testsignal. As described above, the MBR 457 exhibits a dielectric propertywhich modulates the test signal and each MBR 457 will exhibit adifferent dielectric property which will in turn will modulate the testsignal differently. The modulated signals or “signatures” are largelyunique and can be associated with samples having known molecular bindingevents. These stored signals can later be used to identify the molecularstructure in an unknown solution. Molecular structures within the sameclass may exhibit a similar frequency pattern over a common testfrequency range. In this case, the tester is able to identify the classof the unknown molecular structure if the identity of the molecularstructure itself.

[0143] IV. Array Test System and Bio-Assay

[0144] A multitude of bio-assay devices, some examples of which aredescribed in FIGS. 4A-E, may be implemented in an N×M array teststructure to perform high through-put analysis. In this configuration,N×M different binding events may be detected, for instance to enablefast characterization of oligonucleotides such as single nucleotidepolymorphism, individual genes, and longer sequences of the nucleicacides. The number of inputs may be the same as the number of outputs inwhich case M=N, or the number of inputs and outputs may differ.

[0145] The array may be fabricated using conventional photolithographicprocessing to form one or more biosensors on a substrate, such as the0.5 mm² devices described above. Alternatively, the array may befabricated using semiconductor processing techniques, such as SiliconDioxide (SiO₂)or Gallium Arsenide (GaAs) processing. In this embodiment,the array in wafer form may include 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰ bio-assay devices/mm or range anywhere therebetween.

[0146] A. Test System

[0147]FIG. 5 illustrates one possible embodiment of an N×M array testsystem 500 in accordance with the present invention. The test systemincludes a test fixture 600 further described below, a 1×N input switch530, a measurement system 540, a M×1 output switch 550, and a computer560. Measurement system 540 communicates test signals to the testfixture 600 via input test cable 524 a and 1×N input switch 530. Thetest signal is subsequently received from the test fixture via M×1output switch 550 and output test cable 524 b. Computer 560 controls 1×Nin put switch 530, measurement system 540, and M×1 output switch 550 viaa control bus 550.

[0148] In one embodiment, measurement system 540 may consist of theprevious described measurement system 240 or any of the alternativeembodiments described herein. Similarly, input and output test cables524 a and 524 b, control bus 550, and computer 560 may consist of thosepreviously described and/or their alternatives.

[0149] The 1×N input switch 530 routes the test signal from the inputtest cable 524 a to one of the N test fixture signal inputs. The M×1output switch 550 routes the test signal from one of the M test fixtureoutputs to the output test cable. Input and output switches 530 and 550may consist of any switching or multiplexing means which will supportthe propagation of the desired test signal. For instance, input andoutput switches 530 and 550 may consist of low frequency switches (DC to2 GHz), such as those manufactured by Amplifonix, Inc. of Philadelphia,Pa. (www.amplifonix.com). Switches for use at higher frequencies (2-18GHz), such as those manufactured by the General Microwave Corporation ofAmityville, N.Y. (www.generalmicrowave.com) may alternatively beemployed. Connection between bio-assay device and input and outputswitches 530 and 550 may be made using insulated cables, wire bonds, orother conventional interconnection means appropriate for the testfrequency of operation.

[0150] In an alternative embodiment, input and output switches 530 and550 and the bio-assay array form a monolithic integrated circuit. Forinstance, when the bio-assay array is fabricated using GaAssemiconductor processing techniques, input and output switches 530 and550 may consist of integrally formed PIN diodes which are coupled to thebio-assay array. Further alternatively, input and output switches 530and 550 may form an integrated assembly in which the input and outputswitches 530 and 550 are discrete components which are connected (viawire or ribbon bonds) to the bio-assay array. Both alternativeembodiments provide advantages in that the interconnecting structuresare miniaturized or eliminated, thereby reducing or eliminating thesignal loss associated therewith.

[0151] As explained, the bio-assay array may be fabricated in wafer formusing semiconductor processing techniques. In this embodiment, the arraytest system 500 may consist of a wafer probe test station, such as thosemanufactured by Cascade Microtech, Inc. of Beaverton, Oreg.(www.cascademicrotech.com) which includes or is coupled to theaforementioned input and output switches 530 and 550, and computer 560.The wafer probe station utilizes one or more probe cards, each of whichis capable of providing a large number of low loss, low VSWR signalinterconnections to the bio-assay array.

[0152] The probe card(s) may be used to provide N and/or M signalinterconnections to the remotely located input and/or output switches530 and 550, respectively. Alternatively, input and/or output switches530 and 550 may be monolithically fabricated with the bio-assay array,in which case the probe card(s) provides a single input and/outputsignal transition to the measurement system 540. In this latterembodiment, the probe card(s) includes probes for providing switchcontrol voltages to the monolithically formed switches.

[0153] Alternatively or in addition, measurement system 540 may includea Time Domain Reflectometer (TDR) system, such as those optionallyavailable with the aforementioned network analyzers or described in theincorporated patent application entitled: “Method and Apparatus forDetecting Molecular Binding Events,” Ser. No. 09/243,194.

[0154] B. Array Test Fixture

[0155]FIG. 6A illustrates a side view of one possible embodiment of theN×M array test fixture 600 in accordance with the present invention.Similar in construction to the single path test fixture 300 shown inFIG. 3, test fixture 600 includes a top plate 602, bottom plate 604, anda sample cavity 640 which holds the aforementioned reaction vessel 610,bio-assay device 700 (further described in FIG. 7 below), and bottomspacer 630 elements. In the NxM array test fixture embodiment, thedimensions of sample cavity 640 and correspondingly reaction vessel 610and bottom spacer 630 are designed to accommodate the bio-assay device700 which may be larger or smaller than the bio-assay device 300 shownin FIG. 3. Each array element includes a small, monolithically depositedstructure to form a recessed area over the signal path in order to holda portion of the applied sample in electromagnetic communication withthe signal path of each array element. In another embodiment, MEMS(micro-electronic machining systems) technology may be used to fabricatethe sample cavity at the bio-assay device level.

[0156]FIG. 6B illustrates an end view of the N×M array test fixture 600.Test fixture 600 includes N input connectors 660 a ₁ to 660 a _(n) and Moutput connectors 660 b ₁ to 660 b _(m). Test fixture 600 also includesN input transmission lines (not shown) which provide a signal transitionbetween the fixture's N connectors 660 a ₁ to 660 a _(n) and thebioassay's N inputs. Test fixture 600 further includes M outputtransmission lines (not shown) which transition between the bio-assay'sM outputs and the fixture's M output connectors 660 b ₁ to 660b _(m).The input and output transmission lines may be realized as insulatedconductive wires, microstrip, stripline, coplanar waveguide transmissionlines deposited on a dielectric substrate, or other conventionally knownsignal path architectures. The choice of the transmission line'sarchitecture will be influenced by the test frequency band and thebio-assay device's input and output port density.

[0157] C. Bio-Assay Array

[0158] Any or all of the structures shown in FIGS. 4A-4E can be used toform a bio-assay array in accordance with the present invention. Thearray may be fabricated on a discrete piece of dielectric substrate orin wafer form using semiconductor processing techniques. The array mayinclude two or more of the above-mentioned structures on a singledevice, and coupled to diagnostic apparati via any of the standardswitching techniques. Further active elements such as transistors mayalso be used as array elements, as will be further described below.

[0159] One, two, and three dimensional addressing may be used, with anynumber of addresses on the device itself. Each address may be designedto act as a logic gate in which a binary decision is made regardingbinding or some other change in the MBR; to make decisions about threeor more states, such as the shift in frequencies in a band limitedsystem of resonators; or to measure a continuum of properties such asvoltage, phase, frequency, or any of the other parameters as discussedabove.

[0160]FIG. 7A illustrates one embodiment of an integrated bio-assayarray 700 in accordance with the present invention. The integrated array700 is supplied with a test signal via the signal source of measurementsystem 540. The array 700 includes an integrated 1×N input switch andM×1 output switch which are monolithically formed during thesemiconductor fabrication process. The number of inputs may be the sameas the number of outputs in which case M=N, the number of inputs andoutputs may differ.

[0161] The 1×N input switch routes the incoming test signal to thedesired array element. The MBR in the array element modulates the testsignal according to the dielectric properties of the molecular bindingevents which make up the MBR. An M×1 output switch 550 routes themodulated test signal to a detector of the measurement system 540. Ananalyzer of the test system 540 compares the input and modulated testsignals to determine the measured signal response. While each arrayelement is illustrated as a two-port device, those of skilled in the artwill appreciate that one-port or multiple port array elements may beused alternatively.

[0162] As explained above, the array 700 and the input and outputswitches may be fabricated either as discrete components or in waferform and integrated in varying degrees depending upon the application.In the illustrated embodiment, the array 700 and input and outputswitches are monolithically formed on a semiconductor wafer. In anotherembodiment, the input and output switches are monolithically formedseparately from the array 700 and connected via wire or ribbon bonds. Ina further embodiment, input and output switches 530 and 550 and array700 are each discrete units. Those skilled in the art will appreciatethat other arrangements are also possible.

[0163]FIG. 7B illustrates one embodiment of an array element, shown as aseries connected, electronically switched Field Effect Transistor (FET)710. FET 710 may be a Metal Semiconductor Field Effect Transistor(MESFET) fabricated using GaAs processing. Other transistorconfigurations are also possible for instance, High Electron MobilityTransistors (HEMT), heterostructure FETs, homogenous or heterojunctionbipolar transistors, or PN junctions devices such as PIN diodes to namea few. Other active or passive array elements may be used alternativelyor addition to these as well.

[0164] In the embodiment of FIG. 7B, the source and drain terminals 712and 714 of FET 710 are employed as the input and output ports, 711 and715 respectively. The sample is applied over FET 710 such that the MBR716 provides a parallel path between the source and drain terminals 712and 714. FET 710 is designed such that when turned off, it presents adrain to source resistance (R_(ds)) which is much higher than resistancethrough the MBR 716. In this instance, the signal path propagatesthrough the MBR 716 which modulates the test signal. The modulated testsignal is recovered (through a DC blocking capacitor to remove the DCbias) and compared to the input test signal to detect and/or identifythe molecular binding events occurring within the MBR 716. When the FET710 is activated, it provides a much lower R_(ds) compared to theresistance of the MBR 716. In this instance, the MBR 716 is effectivelyswitched out of the signal path and the signal propagates largelyunaffected by it. Thus by simply opening or closing a switch, an arrayelement may be addressed.

[0165]FIG. 7C illustrates a further embodiment of a FET used as an arrayelement which is optically switched. FET 720 is connected similarly toFET 710 described in FIG. 7B and may consist of a photosensitivetransistor, diode or other photosensitive device. The gate junction 722may be illuminated, for instance, with normal sunlight, a laser, a LightEmitting Diode (LED), or other source having a wavelength to which FET720 has a high sensitivity. The incident light activates FET 720 toswitch out the MBR 722. When the FET 720 is deactivated, the test signalpropagates through the MBR 722 and is modulated thereby. The modulatedtest signal is recovered (through a DC blocking capacitor not shown) andanalyzed to determine the presence and/or identity of molecular bindingevents within the MBR 722.

[0166]FIG. 7D illustrates an extension of FIG. 7B and 7C in which two ormore FETs are serially-connected. Array 750 includes a first test path753 along which addressable switches 753 a and 753 c are coupled. In oneembodiment, addressable switches are electronically or opticallycontrolled MESFETs, described above. Array path 753 further includessample regions 753 b and 753 d, each of which provides a parallel signalpaths to the corresponding addressable switches 753 a and 753 c.

[0167] As described above, addressable switches 753 a and 753 c operateto switch in and out the sample regions 753 b and 753 d. Thus, aparticular row is made into a transmission path in which a single assaysite appears as an impedance mismatch. Each assay site can be eitherswitched into the circuit, or switched out of the circuit, as desired.The nature of the impedance mismatch is a function of binding and otherchanges in the MBR. Additional signal paths such as signal path 754 maybe included in the array and cross-strapped to the other paths usingother low loss switches (not shown) to allow the test signal topropagate between signal paths 753 and 754. Input and output switches752 and 755 are used to inject and recover the test signal to/from thearray 750. As those of skill in the art will appreciate, the describedarray may be extended to any number of N×M elements to provide a twodimensional array device.

[0168]FIG. 7E illustrates the circuit equivalent model of the arrayshown in FIG. 7D. The switch impedance Zs is designed to be a closematch with the reference impedance of the signal path Zo, and the assayimpedance Z^(I,J) is designed to be much different than either theswitch or reference impedance. Thus, small changes in the assayimpedance will dominate the electrical properties of any given row, andwill therefore be easily detectable. The exact values for the impedanceswill depend on the design criteria for the particular array, but certaingeneral principles of engineering apply, such as the greatest efficiencyin terms of delivering power to the load (detector) is obtained withmatched-impedance design, and reference impedances are frequently takento be 50Ω.

[0169] In an alternative embodiment, each array element may consist of alogic gate which is capable of occupying one of two possible states,depending on the conditions of gating. As an example, the conditions ofgating may be whether or not a particular binding event has occurred.Such a condition may be the hybridization of nucleic acid material tospecific capture probes on the surface of the device, or a particulardrug-receptor interaction. In any case, the device is engineered so thata binding event or structural change in the MBR triggers the gating.Essentially the modulation of any circuit parameter may trigger thegating; all that is required is to have the necessary hardware andsoftware in place to make the decision as to whether or not the circuitparameter has been modulated.

[0170] As an example, one may monitor a characteristic frequency of agiven system such as a resonant structure. The shift in this frequencyas a result of a particular binding event may serve as the modulationwhich signals the logic state. Any parameter which changes as a functionof binding may be used to trigger logic gate. Such parameters include,but are not limited to: frequency, voltage, current, power, phase,delay, impedance, reactance, admittance, conductance, resistance,capacitance, inductance, or other parameters.

[0171]FIG. 7F illustrates one embodiment of a two-dimensional bio-assayarray 770. As shown, the array 770 includes a first input/output (I/O)axis 772 and a second I/O axis 774 for inputting/outputting testsignals.

[0172] The array is interfaced with conventional external diagnostichardware which is capable of generating and detecting the appropriatefrequency or frequencies, then communicating it to and from the assayarray via a multiplexer, through the ports as illustrated above. Such anexternally supported system may be comprised of any number ofelectromagnetic sources such as vector and scalar network analyzers,time-domain devices like TDR analyzers and other pulsed techniques;utilize any of the detection schemes mentioned herein, including vectorand network analyzers; and use any number of well-known techniques todeliver the signals to and from the assay array via standard andnon-standard multiplexing techniques.

[0173] Generically, such a chip may be fabricated using standardsemiconductor chip approaches. Those of skill in the art will readilyappreciate that such a configuration may be used in a one-port format, atwo port format, or utilize more than two ports.

[0174] V. Applications

[0175] The above described bio-assay, test fixture, and test system maybe used in a number of applications to detect and/or identify particularmolecular binding events occurring within the sample. A few of thepossible applications are described in general below.

[0176] Nucleic Acid Chemistry Application

[0177] The bio-sensors and test systems of the present application maybe used to analyze binding complexes, such as the hybridizationcomplexes formed between a nucleic acid probe and a nucleic acid target.For instance, the bio-assay sensors and test system may be used indiagnostic methods which involve detecting the presence of one or moretarget nucleic acids in a sample, quantitative methods, kinetic methods,and a variety of other types of analysis such as sequence checking,expression analysis and de novo sequencing. One or more of these methodsmay also detect binding between nucleic acids without the use of labels.Certain methods will benefit from utilizing the described bio-assayarrays and test systems which allows for high throughput. Other methodswill benefit from the use of spectral profiles which makes it possibleto distinguish between different types of hybridization complexes. Thesemethods are further described in the incorporated, concurrently filedpatent application entitled “Methods of Nucleic Acid Analysis,” AttyDocket 019501-000600.

[0178] Drug Discovery Application

[0179] The bio-sensors and test systems of the present application maybe used to detect binding events between proteins and a variety ofdifferent types of ligands. The bio-assay sensors and test systems ofthe present invention may be used to screen libraries of ligands toidentify those ligands which bind to a protein of interest, such methodshave particular utility in drug screening programs, for example.Additionally, the bio-assay sensors and test system may be similarlyemployed with diagnostic methods to detect the presence of a particularligand that binds to a known protein, or of a particular protein thatbinds to a known ligand. These methods are further described in theincorporated, concurrently filed patent application entitled “Methodsfor Analyzing Protein Binding Events,” Atty Docket 019501-000700.

[0180]FIG. 8 is an example of the effects of a protein bindingnon-specifically to the dielectric signal path of the bio-assay device450 illustrated in FIG. 4E. A buffer (d-PBS) was initially placed in thedielectric gap region 455 (FIG. 4E) and a baseline insertion lossmeasurement over the frequency range 45 MHz to 40 GHz was taken. Next, asample solution containing urease at high concentration was added andthe urease was allowed to bind to the quartz in the dielectric gapregion 455. The dielectric region 455 was then flushed with d-PBS and asecond insertion loss measurement over the same frequency range wastaken. The second measurement was compared to the first resulting in thechanges in the signal's frequency response, shown in FIG. 8.

[0181] While the above is a complete description of possible embodimentsof the invention, various alternatives, modification and equivalents maybe used to which the invention is equally applicable. Therefore, theabove description should be viewed as only a few possible embodiments ofthe present invention, the boundaries of which is appropriately definedby the metes and bounds of the following claims.

What is claimed is:
 1. A bio-assay test system comprising: a testfixture comprising: a bio-assay device comprising a signal path; and aretaining structure configured to place a sample comprising molecularstructures in electromagnetic communication with the signal path; ameasurement system configured to transmit test signals to and receivetest signals from the signal path at one or more predefined frequencies;a computer coupled to the measurement system configured to control thetransmission and reception of the test signals to and from themeasurement system.
 2. The single path test system of claim 1, whereinthe measurement system comprises a vector network analyzer configured tocompare the magnitude and phase response of the received test signal tothe magnitude and phase response of the transmitted test signal.
 3. Thesingle path test system of claim 2, wherein the test signals comprisesignals in the range of 5 Hz to 300 MHz.
 4. The single path test systemof claim 2, wherein the test signals comprise signals in the range of 45MHz to 40 GHz.
 5. The single path test system of claim 2, wherein thetest signals comprise signals in the range of 33 GHz to 110 GHz.
 6. Thesingle path test system of claim 2, wherein the bio-assay devicecomprises a transmission line.
 7. The single path test system of claim2, wherein the bio-assay device comprises a meandered transmission line.8. The single path test system of claim 2, wherein the bio-assay devicecomprises a ring resonator circuit.
 9. The single path test system ofclaim 2, wherein the bio-assay device comprises a capacitive gapcircuit.
 10. The single path test system of claim 2, wherein thebio-assay device comprises a dielectric signal path.
 11. The single pathtest system of claim 2, wherein the retaining structure comprises anO-ring removeably compressed around a portion of the signal path, theO-ring configured to hold the sample solution in contact with the signalpath.
 12. The single path test system of claim 2, further comprising: aninput connector coupled between the measurement system and a first portof the signal path; and an output connector coupled between themeasurement system and a second port of the signal path.
 13. A bio-assayarray test system, comprising: a test fixture comprising: a bio-assaydevice comprising a plurality of signal paths; and a plurality ofretaining structures configured to place a sample comprising molecularstructures in electromagnetic communication with each of the pluralityof signal paths; a measurement system having at least one output portconfigured to transmit test signals to and at least one input portconfigured to receive test signals from one or more of the plurality ofsignal paths at one or more predefined frequencies; and a computercoupled to the measurement system and configured to control thetransmission and reception of the test signals to and from themeasurement system.
 14. The bio-assay array test system of claim 13,wherein the measurement system comprises one output port and one inputport, and wherein the bio-assay array comprises N input ports coupled tothe plurality of signal paths and M output ports coupled to theplurality of signal paths, the bio-assay system further comprising: a1×N input switch having an input coupled to the measurement systemoutput port and an output coupled to the N signal path input ports; anda M×1 output switch having an input coupled to the M signal path outputports and an output coupled to the measurement system input port. 15.The bio-assay array test system of claim 13, wherein each of theplurality of bio-assay arrays comprises a transmission line.
 16. Thebio-assay array test system of claim 13, wherein at least one of theplurality of bio-assay arrays comprises a meandered transmission line.17. The bio-assay array test system of claim 13, wherein at least one ofthe plurality of bio-assay arrays comprises a ring resonator circuit.18. The bio-assay array test system of claim 13, wherein at least one ofthe plurality of bio-assay arrays comprises a capacitive gap circuit.19. The bio-assay array test system of claim 13, wherein at least one ofthe plurality of bio-assay arrays comprises a dielectric signal path.20. The bio-assay array test system of claim 13, wherein at least one ofthe plurality of bio-assay arrays comprises an electronically switchedtransistor.
 21. The bio-assay array test system of claim 13, wherein atleast one of the plurality of bio-assay arrays comprises an opticallyswitched transistor.
 22. The bio-assay array test system of claim 13,wherein the test signals comprise signals in the range of 5 Hz to 300MHz.
 23. The bio-assay array test system of claim 13, wherein the testsignals comprise signals in the range of 45 MHz to 40 GHz.
 24. Thebio-assay array test system of claim 13, wherein the test signalscomprise signals in the range of 30 GHz to 110 GHz.
 25. A bio-assaydevice, comprising a signal path having an input port and an outputport; and a retaining structure configured to place a sample comprisingmolecular structures in electromagnetic communication with at least aportion of the signal path.
 26. The bio-assay device of claim 25,wherein the signal path comprises a continuous transmission line. 27.The bio-assay device of claim 25, wherein the signal path comprises ameandered continuous transmission line.
 28. The bio-assay device ofclaim 25, wherein the signal path comprises a resonant cavity circuit.29. The bio-assay device of claim 25, wherein the signal path comprisesa capacitive gap circuit.
 30. The bio-assay device of claim 25, whereinthe signal path comprises a dielectric signal path.
 31. A bio-assayarray device, comprising a plurality of signal paths, each having aninput port and an output port; and a respective plurality of retainingstructures configured to place a sample comprising molecular structuresin electromagnetic communication with at least a portion of each of theplurality of signal paths.
 32. The bio-assay array device of claim 31,wherein each signal path comprises an electrically-switched transistor.33. The bio-assay array device of claim 31, wherein each signal pathcomprises an optically-switched transistor.